EP0582317A2 - Synthèse de 1,2-dioxétanes et intermédiaires nécessaire pour cette synthèse - Google Patents

Synthèse de 1,2-dioxétanes et intermédiaires nécessaire pour cette synthèse Download PDF

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EP0582317A2
EP0582317A2 EP19930114829 EP93114829A EP0582317A2 EP 0582317 A2 EP0582317 A2 EP 0582317A2 EP 19930114829 EP19930114829 EP 19930114829 EP 93114829 A EP93114829 A EP 93114829A EP 0582317 A2 EP0582317 A2 EP 0582317A2
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compound
formula
group
alkyl
adamant
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EP0582317B1 (fr
EP0582317A3 (fr
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Brooks Edwards
Irena Y. Bronstein
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Tropix Inc
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Tropix Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/20—Carbocyclic rings
    • C07H15/203—Monocyclic carbocyclic rings other than cyclohexane rings; Bicyclic carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C41/00—Preparation of ethers; Preparation of compounds having groups, groups or groups
    • C07C41/01—Preparation of ethers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/29—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by introduction of oxygen-containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D321/00—Heterocyclic compounds containing rings having two oxygen atoms as the only ring hetero atoms, not provided for by groups C07D317/00 - C07D319/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/08—Esters of oxyacids of phosphorus
    • C07F9/09—Esters of phosphoric acids
    • C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/655—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms
    • C07F9/6551—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring
    • C07F9/65512—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having oxygen atoms, with or without sulfur, selenium, or tellurium atoms, as the only ring hetero atoms the oxygen atom being part of a four-membered ring condensed with carbocyclic rings or carbocyclic ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/6564—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms
    • C07F9/6571—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having phosphorus atoms, with or without nitrogen, oxygen, sulfur, selenium or tellurium atoms, as ring hetero atoms having phosphorus and oxygen atoms as the only ring hetero atoms
    • C07F9/6574—Esters of oxyacids of phosphorus
    • C07F9/65742—Esters of oxyacids of phosphorus non-condensed with carbocyclic rings or heterocyclic rings or ring systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H15/00—Compounds containing hydrocarbon or substituted hydrocarbon radicals directly attached to hetero atoms of saccharide radicals
    • C07H15/26—Acyclic or carbocyclic radicals, substituted by hetero rings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2603/00—Systems containing at least three condensed rings
    • C07C2603/56—Ring systems containing bridged rings
    • C07C2603/58—Ring systems containing bridged rings containing three rings
    • C07C2603/70—Ring systems containing bridged rings containing three rings containing only six-membered rings
    • C07C2603/74—Adamantanes

Definitions

  • This invention relates generally to assay methods in which a member of a specific binding pair can be detected and quantified by means of an optically detectable reaction brought about by the enzymolysis of an enzyme-cleavable group in a 1,2-dioxetane molecule.
  • the invention relates specifically to the production of 1,2-dioxetanes and their intermediates useable in such assay methods.
  • 1,2-Dioxetanes cyclic organic peroxides whose central structure is a four-membered ring containing a pair of contiguous carbon atoms and a pair of contiguous oxygen atoms (a peroxide linkage, are a known, but heretofore seldom utilized, class of compounds. Because of their inherent chemical instability, some 1,2-dioxetanes exhibit chemiluminescent decomposition under certain conditions, e.g., by the action of enzymes, as described in copending, commonly-assigned Bronstein, U.S. patent application Serial No.
  • the concentration of the 1,2-dioxetane (and hence the concentration of the substance being assayed, i.e., the species bound to the 1,2-dioxetane member of the specific binding pair) can be determined.
  • the appropriate choice of substituents on the 1,2-dioxetane ring allows for the adjustment of the chemical stability of the molecule which, in turn, affords a means of controlling the onset of chemiluminescence, thereby enhancing the usefulness of the chemiluminescent behavior of such compounds for practical purposes, e.g., in chemiluminescence immunoassays and DNA probe assays.
  • Such intermediates can be used to prepare substrates which react with singlet oxygen (generated chemically or photochemically) to yield 1,2-dioxetanes of sufficient stability to be useful in subsequent assay techniques based on chemiluminescent dioxetane decomposition
  • This O-alkylation process is general and therefore extendable to other cycloalkyl aryl ketone substrates, which can be synthesized by the reaction of the appropriate secondary cycloalkyl aldehyde with an aryl Grignard reagent, followed by oxidation of the resulting secondary alcohol with Jones reagent.
  • the Grignard reagent is reacted with a secondary cycloalkyl nitrile, followed by acid hydrolysis to form a ketone via an imine salt.
  • starting materials and products contain a functional group attached to a secondary carbon atom of the cycloalkyl system, which in the case of fused polycycloalkyl (e.g., adamantyl) systems is flanked on either side by a bridgehead carbon atom.
  • Yet another object of this invention is to provide novel compositions of matter, such as trisubstituted enolether phosphates, useful as synthetic precursors of 1,2-dioxetanes which dioxetanes decompose enzymatically in an optically-detectable reaction.
  • novel compositions of matter such as trisubstituted enolether phosphates, useful as synthetic precursors of 1,2-dioxetanes which dioxetanes decompose enzymatically in an optically-detectable reaction.
  • T represents a stabilizing group that prevents the dioxetane compound from decomposing before the bond in the labile ring substituent attached to Y is intentionally cleaved, such as an aryl group, a heteroatom group, or a substituted cycloalkyl group having from 6 to 12 carbon atoms, inclusive, and having one or more alkoxy or alkyl substituents containing from 1 to 7 carbon atoms, inclusive, e.g., 4-tertbutyl-1-methyl-cyclohex-1-yl.
  • T may be a cycloalkylidene group bonded to the 3-carbon atom of the dioxetane ring through a spiro linkage and having from 5 to 12 carbon atoms, inclusive, which may be further derivatized with one or more substituents which can be alkyl or aralkyl groups having from 1 to 7 carbon atoms, inclusive, or a heteroatom group which can be an alkoxy group having from 1 to 12 carbon atoms, inclusive, such as methoxy or ethoxy, e.g., 4-tertbutyl-2,2,6,6-tetramethyl-cyclohexyliden-1-yl.
  • the most preferred stabilizing group is a fused polycycloalkylidene group bonded to the 3-carbon atom of the dioxetane ring through a carbon-carbon or a spiro linkage and having two or more fused rings, each having from 3 to 12 carbon atoms, inclusive, e.g., an adamant-2-ylidene or an adamant-2-yl group, which may additionally contain unsaturated bonds or 1,2 fused aromatic rings, or a substituted or unsubstituted alkyl group having from 1 to 12 carbon atoms, inclusive, such as tertiary butyl or 2-cyanoethyl, or an aryl or substituted aryl group such as carboxyphenyl, or a halogen group such as chloro, or a heteroatom group which can be a hydroxyl group or a substituted or unsubstituted alkoxy or aryloxy group having from 1 to 12 carbon atoms, inclusive, such as an eth
  • Y represents a light-emitting fluorophore-forming fluorescent chromophore group capable of absorbing energy to form an excited energy state from which it emits optically detectable energy to return to its original energy state. Any carbon position in Y can be attached to the dioxetane ring.
  • Suitable Y chromophores include:
  • the most suitable Y chromophores are derivatives of benzene or naphthalene:
  • the symbol Z represents hydrogen (in which case the dioxetane can be thermally cleaved via rupture of the oxygen-oxygen bond), a chemically cleavable group such as a hydroxyl group, an alkanoyl or aroyl ester group, or an alkyl or aryl silyloxy group, or an enzyme-cleavable group containing a bond cleavable by an enzyme to yield an electron-rich moiety bonded to chromophore Y, e.g., a bond which, when cleaved, yields an oxygen anion, a sulfur anion, an amine, or a nitrogen anion, and particularly an amido anion such as a sulfonamido anion.
  • This moiety initiates the decomposition of the dioxetane into ketone and ester fragments.
  • electron-rich moieties include oxygen, sulfur, amine, etc. The most preferred moiety is an oxygen anion.
  • suitable enzyme-cleavable groups include enzyme-cleavable alkanoyloxy or aroyloxy groups, e.g., an acetate ester group, or an enzyme-cleavable phosphoryloxy group, oxacarboxylate group, 1-phospho-2,3-diacylgyceride group, D-xyloside group, D-fucoside group, 1-thio-D-glucoside group, adenosine triphosphate analog group, adenosine diphosphate analog group, adenosine monophosphate analog group, adenosine analog group, ⁇ - or ⁇ -D-galactoside group, ⁇ - or ⁇ -D-glucoside group, ⁇ - or ⁇ -D-manno
  • R represents a C1-C20 unbranched or branched, substituted or unsubstituted, saturated or unsaturated alkyl group, e.g., methyl, allyl or isobutyl; a heteroaralkyl or aralkyl (including ethylenically unsaturated aralkyl) group, e.g., benzyl or vinylbenzyl; a polynuclear (fused ring) or heteropolynuclear aralkyl group which may be further substituted, e.g., naphthylmethyl or 2-(benzothiazol-2-yl)ethyl; a saturated or unsaturated cycloalkyl group, e.g., cyclohexyl or cyclohexenyl; a N, O, or S heteroatom containing group, e.g., 4-hydroxybutyl, methoxyethyl, or polyalkyleneoxyalkyl; an a
  • One or more of the formula components T, R, Y or Z can also include a substituent which enhances the water solubility of the 1,2-dioxetane such as a carboxylic acid, sulfonic acid or their salts, or a quaternary amino salt group.
  • a substituent which enhances the water solubility of the 1,2-dioxetane such as a carboxylic acid, sulfonic acid or their salts, or a quaternary amino salt group.
  • At least one of R and Z, and preferably Z, is an enzyme cleavable group, and preferably an enzyme cleavable phosphate ester or glycosidic acetal group.
  • R may be bonded to Y to form a fused ring fluorophore-forming group which is in turn bonded to the 4-carbon atom of the dioxetane through a spiro linkage and which therefore results in an excited lactone fragment upon chemical or enzymatic dioxetane decomposition.
  • the required enol ethers are obtained by intramolecular O-alkylation of fused polycycloalkyl aryl ketone enolates by another substituent, e.g., a toluenesulfonyloxyethyl group, in accordance with the methodology presented herein.
  • Y may also be further substituted with one or more electron withdrawing groups, e.g., perfluoroalkyl having from 1 to 7 carbon atoms such as trifluoromethyl; alkyl or arylsulfonyl such as methylsulfonyl; halogen such as fluoro or chloro; cyano; nitro; alkoxycarbonyl such as -COOEt; alkanoyl such as -COCH3; amidosulfonyl such as -SO2NHAr; or with one or more electron donating groups such as a branched or unbranched alkyl group having from 1 to 7 carbon atoms; an alkoxy or aralkoxy group having from 1 to 30 carbon atoms which may contain fused aromatic or fused heteroaromatic rings which are further substituted with heteroatom containing moieties, e.g., 2-(5-fluoresceinyl)-ethoxy; an aryloxy group having 1 or 2 rings and which may be further
  • 1,2-dioxetanes can be attached to various molecules (e.g., proteins or haptens) or immobilizing supports (e.g., polymer membranes); they can also constitute side chain groups of homopolymers or copolymers.
  • molecules e.g., proteins or haptens
  • immobilizing supports e.g., polymer membranes
  • the method for producing 1,2-dioxetanes according to the present invention comprises the following reaction sequence: wherein R1 can be independently any of the substituents as defined above for R; W is an acid anion such as halide (e.g., chloride); and X and the "R-ylating agent" are as defined below.
  • Step 1 which involves the slow attack of an aromatic Grignard reagent on a nitrile, may be run at reflux in several ethereal solvents such as diethyl ether (34°), THF (67°), or ethylene glycol dimethyl ether (85°).
  • THF e.g., ethylene glycol dimethyl ether
  • Step III is best accomplished in the solvents listed using sodium or potassium hydride or potassium tertbutoxide as the base.
  • This step utilizes reactive alkylating agents to give a kinetic product and can be run conveniently over a temperature range of 0° to 60°C, depending on the "R"-ylating reagent.
  • Dimethyl or diethyl sulfate are particularly useful and inexpensive reagents which display optimum performance between 25° and 60°C.
  • Step IV the phenolic ether cleavage with sodium thioethoxide may be accomplished with soft nucleophiles such as with lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or sodium sulfide in refluxing N-methyl-2-pyrrolidinone are identical in spirit while having other drawbacks from a commercial point of view.
  • soft nucleophiles such as with lithium iodide in refluxing pyridine, sodium cyanide in refluxing DMSO, or sodium sulfide in refluxing N-methyl-2-pyrrolidinone are identical in spirit while having other drawbacks from a commercial point of view.
  • Steps V, VI and VII, as indicated herein, may be performed separately or in one operation.
  • the cyclic phosphorichloridate is utilized not only because of its monofunctionality, chemoselectivity, and enol ether-compatible deprotection mode, but also because, by virtue of pseudo-rotation, it is 106 times more reactive than acyclic versions.
  • an aromatic hydroxyl group is hindered (e.g., a peri position in a polycyclic, aromatic ring system), or if other substituents lower the pKb or nucleophilicity of the enol ether oxyanion, reasonable reaction rates and yields are possible.
  • phosphate triester formation with a Lewis base, or with a preformed alkali metal salt can be effected with all of the phosphorochloridates listed over a temperature range of -30° to 50°C.
  • the ring cleavage with alkali cyanide in DMF or DMSO should be run in a narrow temperature range between 15° and 30°C. In a one pot or in situ mode this is not important and the range widens to 60°C on the high end.
  • phase transfer techniques under catalysis by quaternary ammonium ions or crown ethers to generate an even more reactive "naked" cyanide and thus to utilize organic solvents of higher volatility (e.g., CH2Cl2), facilitating work-up.
  • organic solvents of higher volatility e.g., CH2Cl2
  • the direct use of pure, quaternary ammonium cyanides or sulfinates gives immediate access to phosphate intermediates or products which contain associated gegenions useful in modifying physical properties such as solubility. Such modifications are within the scope of the process parameters disclosed herein.
  • Beta-elimination processes brought to bear on the cyanoethyl substituted phosphate diester may occur under the influence of a wide range of bases.
  • aqueous ammonium hydroxide can be used in vast excess due to its ease of removal at the end of the process.
  • the cleavage can be accomplished over a temperature range of 25° to 100°C.
  • provisions must be made to avoid losses of gaseous ammonia, and thus, a high-pressure vessel or bomb is required.
  • the preferred temperature range is 35° to 55°C, where the phosphate monoester product is quite stable, and where simple glassware outfitted with wired septa can be used as a closed system.
  • Use of alkali metal or quaternary ammonium hydroxides in this step requires close attention to stoichiometry, but as stated above, can provide a variety of mixed gegenion phosphate salts.
  • sensitized photooxygenation is a particularly convenient and forgiving process when reactive olefins are used as substrates.
  • sensitizing dyes may be used to advantage, with chlorinated hydrocarbons comprising a preferred class of solvents. Reactions are rapid over a temperature range of -78° to 25°C.
  • step VI alkyl cleavage with a nucleophilic acidifying anion such as CN ⁇ or organic sulfinate ion
  • step VII deprotection via a beta-elimination reaction
  • the cation, M+, in the salt used in step VI and the cation, M+, in the base used in Step VII can be an alkali metal (e.g., Na+), ammonium, or a C1 - C7 alkyl, aralkyl, or aromatic quaternary ammonium cation, (NR4)+ (wherein R4 can be any or all of an alkyl, e.g., ethyl, aralkyl, e.g., benzyl, or form part of a heterocyclic ring system, e.g., pyridinium), so that the products of steps VII and VIII would be as follows:
  • the quaternary ammonium cation can be connected through one of its quaternizing groups to a polymeric backbone, as follows: or can itself be part of a polyquaternary ammonium salt.
  • M+ can also be a fluorescent onium moiety such as a substitute
  • an alkylating agent or in more general terms consistent with the definition of R, an "R-ylating agent”
  • R-sulfate toluenesulfonate
  • the invention further provides a process for producing a compound having the formula: wherein T, R, and Y are as defined above, comprising reacting a compound having the formula: with wherein X is an electronegative leaving group such as halogen (e.g., chloro), in the presence of a Lewis base such as a tertiary amine (e.g., triethylamine) dissolved in an aprotic organic solvent, such as an aromatic liquid (e.g., benzene, toluene), and ether (e.g., glyme, diglyme) or a cyclic ether (e.g., tetrahydrofuran (“THF”)).
  • a Lewis base such as a tertiary amine (e.g., triethylamine) dissolved in an aprotic organic solvent, such as an aromatic liquid (e.g., benzene, toluene), and ether (e.g., glyme, dig
  • the analogous halophosphites i.e., XPO2(CH2)2
  • XPO2(CH2)2 can be used with subsequent oxidation and irradiation to form the dioxetane directly.
  • the invention provides a process for producing compounds having the formulas: wherein T, R and Y are as defined above, R5 can be independent of any of the substituents described above for R, and R2 and R3 are each independently cyano, ortho- or para-nitrophenyl, ortho, para- or ortho, ortho'-dinitrophenyl, comprising reacting a compound having the formula: with wherein X is as defined above, in the presence of a Lewis base such as a tertiary amine (e.g., a trialkylamine) in an aprotic organic solvent such as an aromatic liquid (e.g., benzene or toluene), an ether (e.g., glyme, diglyme) or a cyclic ether (e.g., THF).
  • a Lewis base such as a tertiary amine (e.g., a trialkylamine)
  • an aprotic organic solvent such as an aromatic liquid (e.g., benz
  • halophosphates As an alternative to the use of halophosphates, the analogous nor-oxy compounds (i.e., halophosphites) can be used, followed by oxidation at the phosphorous, deprotection and photooxidation to the dioxetane.
  • halophosphites i.e., cyclic phosphite
  • dioxetane formation and oxidation at the phosphorous can occur simultaneously in the presence of 3O2/1O2 mixtures found in the photooxidation reaction.
  • the oxidation described above is effected photochemically by treating the olefin with singlet oxygen (1O2) in the presence of light.
  • 1O2 adds across the double bond to form the dioxetane as follows:
  • the reaction is preferably carried out at or below 0°C in a halogenated solvent, e.g., methylene chloride.
  • 1O2 can be generated using a photosensitizer.
  • photosensitizers polymer-bound Rose Bengal (commercially known as Sensitox I and available from Hydron Laboratories, New Brunswick, N.J.) and methylene blue (a well-known dye and pH indicator) or TPP (see Example 17 below) can be used.
  • the present invention also comprises a process for producing a compound of the general structure: wherein R, T and Y are as defined above, and Z is a D-sugar molecule linked to Y via a glycosidic linkage, by first reacting a component of the following general structure: wherein Y is a phenyl or naphthyl group, with a tetra-O-acetyl-D-hexopyranosyl halide to produce an intermediate of the following general structure:
  • Y is a phenyl or naphthyl group
  • a tetra-O-acetyl-D-hexopyranosyl halide to produce an intermediate of the following general structure:
  • the acetate protective groups are removed by hydrolysis to produce the following general structure:
  • the photochemical oxidation reaction described above is applied to the above intermediate to produce as a product: wherein T and X are described above, Y is a fluorophore such as a phenyl or naphthyl moiety, and Z is a sugar linked to Y via an ⁇ or ⁇ glycosidic bond.
  • the dioxetanes of the invention provide a method for generation of light in an optically detectable assay method to determine the presence or concentration of a particular substance in a sample.
  • assays include immunoassays to detect antibodies or antigens (e.g., hormones such as ⁇ or ⁇ -hCG, TSH, LH, etc., cancer-associated antigens such as AFP and CEA) (enzyme-immunoassay); enzyme assays (e.g., alkaline phosphatases and ⁇ - or ⁇ -D-galactosidases); chemical assays to detect cations, e.g., potassium or sodium ions; and nucleotide probe assays to detect, e.g., viruses (e.g., HSVI, HTLV III, hepatitis virus, cytomegalovirus), or bacteria (e.g., E . coli )).
  • viruses e.g., HSVI,
  • the enzyme capable of cleaving group Z of the dioxetane is preferably bonded to a substance (i.e, a substance that binds specifically to the detectable substance), e.g., an antigen, antibody, or nucleic acid probe, respectively.
  • a substance i.e, a substance that binds specifically to the detectable substance
  • Conventional methods e.g., carbodiimide coupling, are used to bond the enzyme to the specific affinity substance; bonding is preferably through an amide linkage.
  • assays are performed as follows.
  • a sample suspected of containing a detectable substance e.g., antigen
  • a buffered solution containing an enzyme bonded to a substance having a specific affinity for the detectable substance e.g., antibody
  • the resulting solution is contacted with a solid phase, e.g., antibody-binding beads, to which another substance having the specific affinity, e.g., antibody, is bound.
  • excess enzyme which is bound to be substance with specific affinity is then washed away, and a 1,2-dioxetane (substrate) having a group Z that is cleavable by the enzyme portion is added.
  • the enzyme cleaves group Z, causing the dioxetane to decompose into ketone and ester moieties; chromophore Y bonded to the ester is thus excited and luminesces.
  • Luminescence is detected using, e.g., a cuvette or camera luminometer, as an indication of the presence of the detectable substance in the sample. Luminescence intensity is measured to determine the concentration of the substance.
  • an assay for the enzyme involves adding 1,2-dioxetanes to the enzyme-containing sample, and detecting the resulting luminescence as an indication of the presence and the concentration off the enzyme.
  • the precipitate was separated by filtration, washed with ether, and dried to obtain 29 g of the ketenimine salt as a light, buff-colored, non-hygroscopic powder containing some residual magnesium.
  • the salt was suspended in a mixture of 90 ml of ethanol and 90 ml of concentrated hydrochloric acid and refluxed for 3 hours, during which time the mixture became considerably thinner. After cooling in an ice bath, the resulting solid was broken up, separated by filtration, washed to neutrality and dried to obtain 13.65 g (93% yield based on 2-cyanoadamantane) of the light gray ketone (m.p. 111-114°C).
  • a quantity (11.3 g, 0.042 mol) of 3-methoxyphenyl adamant-2-yl ketone obtained according to Example 1 was suspended in 90 ml of molecular sieve-dried (3 ⁇ ) dimethylsulfoxide (DMSO). Heat was applied to dissolve the suspended solid. Upon cooling to room temperature with stirring, a fine suspension was formed. Potassium tertbutoxide (8.5 g, 0.070 mol) was added under an argon atmosphere. After 5 minutes, a nearly homogenous orange solution resulted, which was placed in a water bath at 50°C. Dimethyl sulfate (4 ml, 0.042 mol) was added dropwise by syringe over a period of 10 minutes.
  • DMSO dimethylsulfoxide
  • the aqueous layer was separated and extracted with 75 ml of ethyl acetate.
  • the organic extract was washed with four 100 ml portions of water, then with saturated NaCl solution (100 ml), and quickly dried over Na2SO4.
  • the solution was filtered and concentrated to an oily substance which was then triturated with 50 ml of hexanes.
  • a solid separated Upon removal of the solvent on a rotary evaporator, a solid separated, which was then triturated with cold hexanes, filtered and washed with hexanes.
  • the crude, off-white phenolic product (13 g) was recrystallized from 5% MeOH in CH3CN to yield 10 g of colorless prismatic crystals (m.p.
  • the gas flow was adjusted so as to just maintain a uniform suspension of the solid-phase sensitizer. After 25 minutes of irradiation time, the U.V. (260 nm) absorption of the starting material disappeared. The light yellow solution was filtered, evaporated, and reconstituted with 10 ml water. The aqueous sample was filtered through a 0.45 micron nylon filter and chromatographed on a reverse phase, C18 preparative HPLC column using a water/acetonitrile gradient. The fractions showing weak U.V. absorption at 277 nm were combined and lyophilized to provide the dioxetane as a white, cotton-like, hygroscopic solid.
  • AMPPD Na+NH4+ salt did not exhibit a melting point. Instead, subliming vaporization occurred between 145° - 150°C. A solid residue remained which partially decomposes but did not melt below 270°C.
  • Methoxy (3-phosphoryloxyphenyl)methylene adamantane sodium ammonium salt (3.3g) was dissolved in 15 ml of water containing a drop of pyridine. The solution was slowly run over a 3 cm x 25 cm column of Amberlite IR 120 (plus) ion exchange resin in the pyridinium salt form (Aldrich Chemical Co.). Upon elution with distilled water, the fractions showing absorbance at 260 nm. were combined and lyophilized. A portion of the resulting mono pyridinium salt (1 g, 2.3 mmol) was dissolved in 100 ml of CHCl3 (dried over Al2O3).
  • the resulting solution was placed in a large cylindrical tube and treated with 5, 10, 15, 20-tetraphenyl-21H, 23H-porphine (2 mg. in 1 ml of CHCl3).
  • the homogeneous green solution was cooled to 0° and pre-saturated with oxygen gas via a sparger tube.
  • the mixture was irradiated Under constant O2 flow in a silvered Dewar flask which also contained a cooled immersion well surrounding a 250 watt sodium vapor lamp which was filtered by a single sheet (5 mil) of DuPont Kapton® polyimide film.
  • the temperature in the Dewar remained at 0° - 5°C during a 12 minute irradiation.
  • the solvent was removed in vacuo followed by the addition of 100 ml of distilled water containing 500 mg of NaHCO3.
  • the resulting light pink solution was cooled and filtered through a 0.45 micron Teflon® membrane.
  • the resulting aqueous solution of dioxetane was subjected to a CH3CN-H2O gradient on a polystyrene chromatography column, followed by a second pass with a CH3CN - H2O gradient.
  • the resulting solution which was free of inorganic salts, was lyophilized to produce 800 mg of a granular, faintly yellow, white solid.
  • the sample was then irradiated with a 250 watt, high pressure sodium lamp at a distance of 3 inches (7.62 cm) from the outer edge of the flask. The disappearance of the band at 260 nm in the UV spectrum was monitored over a 3-hour period. After removal of the sensitizer, the slightly yellow solution was concentrated and chromatographed on a reverse phase, C18 preparative HPLC column using 60% acetonitrile/water to 100% acetonitrile gradient. Evaporation of the appropriate fractions provided the dioxetanes as an oil.
  • I.R. (CHCl3, cm ⁇ 1): 3590, 3360 (broad), 3000, 2920, 2855, 1597, 1588, 1448, 1290, 1175, 1066, 954, 870, 854, 710.
  • Analytical HPLC (UV detector at 254 nm) showed complete dioxetane formation after irradiating 10.5 minutes. The reaction was also followed by UV spectroscopy with absorption at 255nm due to the conjugated vinyl group disappearing upon photoxygenation. The dioxetane showed one major UV absorption at 230nm. After evaporating the chloroform at 0°C, the residue was dissolved in ice water, passed through a 0.46 ⁇ filter, and separated by preparative HPLC on a polystyrene column with an acetonitrile/water gradient. The fractions were frozen and lyophilized at 0°C, yielding 12.1 mg (60%) of the disodium phosphate dioxetane as a white, fluffy powder.
  • the crude product were separated into two fractions by the filtration through a short silica gel column, eluting with 25 - 40% EtOAc in hexanes.
  • the less polar mixture (1.05 g) contained mainly the enol ether starting material and a small amount of methoxy(3-acetoxyphenyl)methyleneadamantane.
  • the Rf values on TLC (Whatman K5F; 30% EtOAc in hexane) were 0.64 and 0.76 respectively. This mixture could be treated with NaOMe in MeOH to regenerate the starting material, which was recycled.
  • Ice water was pumped through the apparatus to maintain the sample temperature below 10°C.
  • the solution was irradiated for 10 minutes under constant O2 flow, during which time the U.V. absorption at 261nm (CHCl3) of the starting material disappeared and a new peak at 272nm with a shoulder at 278nm appeared.
  • the solvent was evaporated at low temperature and the residue was triturated with 30% CH3CN in H2O.
  • the aqueous sample was filtered through a 0.45 micron nylon filter and chromatographed on a reverse phase preparative HPLC column using a water-acetonitrile gradient. After lyophilization the dioxetane was collected as a white, cotton-like powder in good yield.

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EP93114829A 1987-12-31 1989-01-03 Synthèse de 1,2-dioxétanes et intermédiaires nécessaire pour cette synthèse Expired - Lifetime EP0582317B1 (fr)

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* Cited by examiner, † Cited by third party
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EP0757248A2 (fr) * 1995-07-31 1997-02-05 Board Of Governors Of Wayne State University Méthodes analytiques à base de la chimie capsulaire utilisant la luminescence de dioxétane
EP0757052A1 (fr) * 1995-07-31 1997-02-05 Board Of Governors Of Wayne State University Composés de dioxétanes-1,2 substitués ayant une solubilité dans l'eau accrue et compositions de dosage

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Publication number Priority date Publication date Assignee Title
US5004565A (en) * 1986-07-17 1991-04-02 The Board Of Governors Of Wayne State University Method and compositions providing enhanced chemiluminescence from 1,2-dioxetanes
US4978614A (en) * 1988-10-26 1990-12-18 Tropix, Inc. Method of detecting a substance using enzymatically-induced decomposition of dioxetanes
US4956477A (en) * 1987-12-31 1990-09-11 Tropix, Inc. Synthesis of 1,2-dioxetanes
US4952707A (en) * 1988-06-30 1990-08-28 Tropix, Inc. Enzymatically-cleavable chemiluminescent fused polycyclic ring-containing 1,2-dioxetanes
US4931569A (en) * 1988-09-14 1990-06-05 Tropix, Inc. Purification of stable water-soluble dioxetanes
US6022964A (en) * 1989-07-17 2000-02-08 Tropix, Inc. Chemiluminescent 1,2-dioxetanes
US5362916A (en) * 1989-09-06 1994-11-08 Tropix, Inc. Synthesis of mercaptaryl or hydroxyaryl enol ether alkali metal salts
EP0441948A4 (en) * 1989-09-06 1992-06-03 Tropix, Inc. Synthesis of stable, water-soluble chemiluminescent 1,2-dioxetanes and intermediates therefor
US5225584A (en) * 1989-09-06 1993-07-06 Tropix, Inc. Synthesis of stable water-soluble chemiluminescent 1,2-dioxetanes and intermediates therefor
WO1994003812A1 (fr) * 1992-07-31 1994-02-17 Syntex (U.S.A.) Inc. Matrices chimioluminescentes photoactivables
KR0148439B1 (ko) * 1994-03-11 1998-11-02 마사까쓰 마쓰모또 1,2-디옥세탄 유도체
US5783381A (en) * 1995-10-19 1998-07-21 Tropix, Inc. Chemiluminescent 1,2-dioxetanes
US6660529B2 (en) * 1998-07-28 2003-12-09 Pe Corporation Heteroaryl substituted benzothiazole dioxetanes
US6355441B1 (en) * 1998-07-28 2002-03-12 Tropix, Inc. Benzothiazole dioxetanes
CA2370520A1 (fr) * 1999-04-16 2000-10-26 Komandoor Elayavalli Achyuthan Procede de detection de virus utilisant des enzymes codees par des virus et des substrats chimioluminescents
WO2002008754A1 (fr) * 2000-07-26 2002-01-31 Chemometec A/S Epreuve biologique liee a une enzyme a resolution spatiale
EP1342724B1 (fr) * 2002-03-08 2016-01-06 Tosoh Corporation Dérivés de 1,2-dioxétannes et les réactifs les utilisant
JP4259229B2 (ja) 2003-08-28 2009-04-30 東ソー株式会社 １，２−ジオキセタンの化学発光方法および化学発光用組成物
JP2012140330A (ja) * 2010-12-28 2012-07-26 Tosoh Corp 水溶性リン酸エステルの精製方法
ES2731833T3 (es) 2012-09-10 2019-11-19 Principia Biopharma Inc Compuestos pirazolopirimidínicos comos inhibidores de cinasas
US11198785B2 (en) 2017-10-13 2021-12-14 Mitsubishi Engineering-Plastics Corporation Polycarbonate resin composition
JP7007862B2 (ja) * 2017-10-31 2022-01-25 アークレイ株式会社 化学発光試薬組成物及び化学発光試薬キット

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0254051A2 (fr) * 1986-07-17 1988-01-27 The Board Of Governors Of Wayne State University Composés 1,2-dioxétane chimialuminescents

Family Cites Families (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2909557A (en) * 1955-09-07 1959-10-20 Bayer Ag Thiophosphoric acid esters and their production
DE1810343U (de) 1960-02-29 1960-04-28 Mak Maschb Kiel G M B H Vorrichtung zum befestigen von achshaltertegen am rahmen von schienenfahrzeugen.
NL6716437A (fr) * 1967-12-02 1969-06-04
US3711556A (en) * 1968-06-13 1973-01-16 Du Pont 2-(bis(p-substituted phenyl)methylene)adamantanes
GB1497109A (en) * 1975-10-29 1978-01-05 Beecham Group Ltd Naphthalene derivatives
JPS57159499A (en) * 1981-03-26 1982-10-01 Fuji Photo Film Co Ltd Substrate for determining alkaline phosphatase and determining method
JPS62176941A (ja) * 1986-01-28 1987-08-03 Sumitomo Electric Ind Ltd 光フアイバ
JPS639377A (ja) * 1986-06-30 1988-01-16 Sony Corp マルチチヤンネル録画装置
US4983779A (en) 1986-07-17 1991-01-08 The Board Of Governors Of Wayne State University Process for the preparation of vinyl ethers
JPH0521918A (ja) * 1991-07-11 1993-01-29 Nippon Cement Co Ltd セラミツクス回路基板用導電パターン形成用粉末組成物
JP3217396B2 (ja) * 1991-08-16 2001-10-09 オリンパス光学工業株式会社 顕微鏡の粗微動機構
JPH0723989B2 (ja) * 1992-08-03 1995-03-15 ビソーディスプレイ株式会社 スクリーン装置
JPH0733346A (ja) * 1993-07-20 1995-02-03 Hitachi Building Syst Eng & Service Co Ltd エレベータの着床制御装置
DE4423811C1 (de) * 1994-07-06 1996-01-18 Fraunhofer Ges Forschung Hydrolysierbare und polymerisierbare Silane, Verfahren zu deren Herstellung und deren Verwendung

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0254051A2 (fr) * 1986-07-17 1988-01-27 The Board Of Governors Of Wayne State University Composés 1,2-dioxétane chimialuminescents

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
TETRAHEDRON LETTERS. vol. 28, no. 44 , 1987 , OXFORD GB pages 5319 - 5322 RUGGERO CURCI ET AL 'Synthesis of 1,2-dioxetanes via electron-transfer oxygenation' *
TETRAHEDRON LETTERS. vol. 28, no. 9 , 1987 , OXFORD GB pages 935 - 938 A. PAUL SCHAAP ET AL 'Chemical and enzymatic triggering of 1,2-dioxetanes.1:aryl esterase-catalyzed chemiluminescence from a naphthyl acetate-substituted dioxetane' *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0757248A2 (fr) * 1995-07-31 1997-02-05 Board Of Governors Of Wayne State University Méthodes analytiques à base de la chimie capsulaire utilisant la luminescence de dioxétane
EP0757052A1 (fr) * 1995-07-31 1997-02-05 Board Of Governors Of Wayne State University Composés de dioxétanes-1,2 substitués ayant une solubilité dans l'eau accrue et compositions de dosage
EP0757248A3 (fr) * 1995-07-31 1997-07-23 Univ Wayne State Méthodes analytiques à base de la chimie capsulaire utilisant la luminescence de dioxétane

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ES2012941A6 (es) 1990-04-16
JPH02724A (ja) 1990-01-05
EP0368946A1 (fr) 1990-05-23
DE68929060T2 (de) 1999-12-09
JPH0521918B2 (fr) 1993-03-25
JP3025280B2 (ja) 2000-03-27
JP3280342B2 (ja) 2002-05-13
EP0582317B1 (fr) 1999-08-25
JPH0731201B2 (ja) 1995-04-10
WO1989006226A1 (fr) 1989-07-13
JPH0733346B2 (ja) 1995-04-12
IL88798A0 (en) 1989-07-31
ZA889659B (en) 1989-09-27
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JPH0640996A (ja) 1994-02-15
IL88798A (en) 1996-01-31
JPH0476997B2 (fr) 1992-12-07
GR1000118B (el) 1991-06-28
EP0582317A3 (fr) 1994-02-23
EP0368946B1 (fr) 1994-09-14
JPH11335342A (ja) 1999-12-07
AU2945189A (en) 1989-08-01
JPH02502916A (ja) 1990-09-13
JPH0641158A (ja) 1994-02-15
DE68929060D1 (de) 1999-09-30
DE68918230T2 (de) 1995-03-02
EP0368946A4 (fr) 1990-05-14
US6417380B1 (en) 2002-07-09
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